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Two Methods for Pre-Hybridization Array QC

Learn about two methods for pre-hybridization array quality control: reflection imaging and ethidium bromide staining. These methods help verify the quality and quantity of spots on the array before applying your probe.

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Two Methods for Pre-Hybridization Array QC

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  1. Two Methods for Pre-Hybridization Array QC Be certain of your spots before you apply your precious probe

  2. Array QC: Definitions and Motivations • Array Quality Control • A process to verify that there are enough good spots on the array to support the subsequent hybridization experiment • “Enough” spots, “Good” spots need to be specified by the researcher depending on type of array and type of experiment • Might be done on a 1 array-per-spotting-batch auditing basis or… • Might be done on every single spotted array • Motivations • Unique, irreplaceable RNA sample: needle biopsy of tumor • Labeled probe mixture is costly,  $100 U.S.

  3. Advantages: Quick, Easy, & Free No reagents at all! Any ScanArray can do it Limitation: Does not precisely measure the quantity of DNA Verifies that the spotter delivered a droplet Must be done prior to washing Analogous to a microscope inspection What it does: detects dried salt in microarray spots (pre-washing) How it works Salt crystals in dried spotting buffer scatter and reflect light A neutral-density filter allows a scanner to image the reflected scattered laser light Method 1: Reflection Imaging Fluorescence Detection Emission filter PMT Laser excitation Fluorescence emission Dye emits fluorescence

  4. Some Reflection Images for Array QC A pre-wash, pre-hyb image from CCD camera on a lab microscope The same array: reflection image from a ScanArray

  5. Reflection Imaging: A Short Protocol • Create custom RedReflection “fluorophore” in ScanArray software* • Red 633 laser (Laser 1) • Neutral density filter (Filter 12) • Print your arrays with 3x SSC or similar high-salt buffer • Re-hydrate & snap dry • Scan using Red Reflection fluor, 100% laser, 80% PMT * ScanArray Express software has Red Reflect fluor pre-defined

  6. QuantArray Determines Spot / No Spot • Using QuantArray’s spot signal/noise feature it is easy to identify missing spots • Spot finding and quantitation works just as with a hybridized array

  7. At least 2 orders of magnitude difference Can then look up the gene IDs of the missing spots and determine whether or not to use the array Signal-to-noise metric in spot quality section is a clear indicator of spot / no spot It’s Easy to Find Missing Spots in the Spreadsheet }

  8. Advantages: Quantitative assessment of amount of immobilized DNA in each spot after washing: This is what you really want to know EtBr washes out and doesn’t affect DNA assays Disadvantages Requires blue light excitation to scan A lot more work than reflection imaging What it does: quantifies the amount of DNA bound to the substrate in each spot How it works Print, UV attach, wash & dry Stain with EtBr; it binds only to the DNA. Scan: 488 nm excitation, 614 nm emission with ScanArray 5000 Quantitate Wash out the EtBr Method 2: Ethidium Bromide Staining EtBr Spectral Data Courtesy Molecular Probes

  9. Reflection + EtBr Stained Example • Printed dilutions of cDNAs: 100, 80, 60, 40, 20 10, 1, 0 g/ml • First, a reflection scan to look at the spots before treatment • Then stained with EtBr and scanned with 488 nm ex. / 614 nm emission Reflection image EtBr image

  10. A Closer Look: EtBr staining quantifies the amount of immobilized DNA 80 g/ml 100 g/ml 60 g/ml 40 g/ml 20 g/ml 10 g/ml 0 g/ml 1 g/ml

  11. Wash It, Then the EtBr is All Gone After ethanol wash, same scan settings After boiling to denature, same scan settings

  12. EtBr Staining: A Short Protocol E. Kawasaki, S. Patel, Oct. 2001 • Steam re-hydrate printed arrays, snap dry • Optional reflection scan 633 laser / ND filter • Fix DNA to substrate (UV 120 mJ/cm2 or 80C 1 hr) • Incubate arrays at 55C 15 min in TES (TE + 0.1% SDS) • Wash 2x, 1 min at room temp in TE • Stain by immersing in 0.1 g/ml EtBr 5 min, room temp • Wash 2x, 2 min in TE. Dry. • Scan with 488 nm excitation, 614 nm emission • Wash out EtBr by soaking/shaking substrate in 75% ethanol, 25% TE for 15 – 30 min OR 1% SDS in 1x TE for 5 min, 1x TE 5 min, ddH20 2 min • Rinse with TE, proceed to Step 11 or store for later use • Boil to denature just before hybridizing

  13. More To Come… • Formal Technical Notes and Application Notes from PerkinElmer will be available later this year • Detailed protocols, sample data • Other spot QC methods are under development at PerkinElmer Life Sciences • Other DNA stains • End-labeling a fraction of the PCR primers before making PCR products to spot • Hybridizing labeled 9-mers (AlexaFluor-labeled Panomers from Probes.) • And…

  14. Thank You! Acknowledgements: Ernie Kawasaki, PhD; Sonal Patel; Jay Gehrig (Packard BioChip) References: Battaglia, C., Salani, G., Consolandi, C., Rossi L., and DeBellis, G. (2000) Analysis of DNA Microarrays by Non-Destructive Fluorescent Staining Using SYBR Green II. BioTechniques 29:78-81 Khitrov, G. (2001) Use of Inexpensive Dyes to Calibrate and Adjust Your Microarray Printer. BioTechniques 30:748 Ethidium Bromide product information, Molecular Probes website http://www.probes.com/servlets/product?region=Select+Region&item=1305

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